RNA Translation

RNA Translation: RNA makes Protein

In principle:
Translation of messenger RNA (mRNA) takes place on ribosomes,
which include ribosomal RNA (rRNA),
with the help of transfer RNA (tRNA)

Structure of rRNA & tRNA

ribosomal RNA (rRNA)
       rRNA + ribosomal protein ribosomes [iG1 6.09]
        Structure of rRNA: stems & loops [iG1 6.05]
             stems: double-stranded (dsRNA)
             loops: single-stranded (ssRNA)

        Structure of eukaryotic ribosomes [iG1 6.04] [iG1 6.14b]
           Large Subunit (LSU) = 60S = 28S rRNA + 5.8S + 5S rRNA + 50 proteins
         Small Subunit (SSU) = 40S = 18S rRNA + 33 proteins
                                                 = 80S monosome

A site (Aminoacyl), P site (Peptidyl), & E site (Exit) [APE complex]

transfer RNA (tRNA)
       the adaptor molecule: ~30 tRNA types
       2-dimensional 'cloverleaf' model [iG1 6.10]
            small: 75 ~ 90 nucs
       stems & loops
            D-loop & TC-loop ( = pseudo-uridylic acid)
                   tRNA characterized by 2^omodified bases [iG1 6.19]
            amino-acceptor stem
                    3' - ~~~~ CACCA - 3'
                    5' -~~~~ G    - 5'
            anticodon loop
                   specificity of tRNA for mRNA determined by 3-ribonucleotide sequence

3-dimensional structure is an "L" [iG1 6.12]
D- & TC-loops fold back on each other

Charged tRNA: aminoacyl synthetase_(x) forms ester linkage between
       3'-A of amino-acceptor stem of tRNA_(x) joined to COOH of amino acid_(x)
       ~20 synthetase types 'recognize' correct anticodon loop
       isoacceptance:
           one-to-one correspondence between synthetase & amino acid

RNA Translation: Protein Synthesis

Ribosomes "read" mRNA & assemble polypeptide according to the Genetic Code

Initiation at start codon (AUG)
              SSU binds at Shine-Delgarno sequence (-6 nucs)
              Initiation Complex consists of mRNA, ribosome, & tRNA
         Multiple complexes form on a single mRNA: polysome (polyribosome)

tRNA_fmet always added first [N-formyl-methionine in prokaryotes]

In simplified form,

   5'-AUG-3' codon in mRNA
      |||
   3'-UAC-5' anticodon in tRNA

5'-CAU-3' if anticodon is written 5' 3'

Elongation: addition of amino acids according to Genetic Code

Amino acids are joined via peptide bonds (see next section)
         Think of mRNA as stationary: ribosome moves along it 5'3'
                peptidyl (P) site on 5' end,
                aminoacyl (A) site on 3' end

        first AUG codon [for met] is in P site
            second UUC codon enters A site
                corresponding tRNA_phe enters A site

        peptide bond formed between fmet & phe
           P site amino acid transferred to A site amino acid
              uncharged tRNA released from P site (passes to E site)
               amino end of fmet remains unchanged ]

and so on ...
growing polypeptide in P site joins single amino acid in A site

   "Wobble": pairing of codon / anticodon goes 5'3' on codon [iG1 7.25, 26]
                      last position can miss-pair with either purine / pyrimidine
                            Fewer tRNA species needed:
                       Ex.: three tRNA_ser species for six codons

tRNA Anti-codon	Alternative Serine mRNA codons
3'- AG G -5'	5'- UC C / U -3'
3'- AG U -5'	5'- UC A / G -3'
3'- UC G -5'	5'- AG C / U -3'

Termination: release of polypeptide

^mRNA^{+ tRNA}(aa_n-...-aa₃-aa₂-aa₁)

^here^{: mRNA + tRNA}(lys-pro-gly-phe-fmet)

      stop codon (UAG, UAA, or UGA) enters A site
                no corresponding tRNA:
                release factor cleaves polypeptide from terminal tRNA_n
                polypeptide product is:   lys - pro - gly - phe - fmet

A talkie animation of transcription & protein synthesis

Griffiths et al. (1996) Fig. 13-7 is a nice summary (HOMEWORK #11)

Bioinformatics of DNA, mRNA, & Protein

5'- G T A A T C C T C - 3' DNA sense strand

5'- G U A A U C C U C - 3' mRNA

N - val - ile - leu - C protein

This is a logical, not a biochemical, relationship:
        Because mRNA is transcribed from the template strand,
        it "looks like" the sense strand (except for 'U').
        The information content of the DNA sense strand and mRNA are identical

Protein sequences can be read directly from DNA:
         Read the sense strand in the 5'3' direction,
         Substitute 'T' for 'U' in the code table [or in your head]
       Computer programs (Chromas, Sequencher, etc.) do this automatically

There are three reading frames on either strand
X two 5'3' strands six possible ways to read dsDNA
Open Reading Frames suggest protein sequences

Deducing protein sequences from random DNA sequences is a major research activity
Bioinformatics: extraction of information from large macromolecular datasets

The following clues are useful:
         Remember that all prokaryotic coding sequences:
            are read only in the 5'3' direction
            begin with a "start" (AUG) codon
          end with a "stop" (UAG, UAA, or UGA) codon.
             Ex.: a typical exam problem is to identify a polypeptide of six amino acids from a dsDNA molecule

          But: in real life research, cloned eukaryotic DNA
                may not have start or the stop codon for a complete protein,
                   [and not all AUG codons are 'start' codons]
                and may be include an intron with one or more 'stop' triplets .

Do not assume that a dsDNA molecule is read from left to right, on the top strand

Homework #12:

Practice DNA "Translation" problems [PDF download version]

There's an App for that: RandORF for 'translation' problems